Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress

Abstract

Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units/kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1--10 units/ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units/ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1/protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported.

Figures

Figure 1

EPO inhibits programmed cell death in the ischemic penumbra after middle-coronary artery (MCA) occlusion in rats. EPO administration at the onset of ischemia resulted in an almost complete protection from apoptosis (as assessed by TUNEL labeling performed 24 h later). Representative sections illustrating cells undergoing apoptosis within the ischemic penumbra in animals treated with saline (control) or r-Hu-EPO (5,000 units/kg of body weight) given i.p. at the onset of a reversible 1-h occlusion of the contralateral carotid artery. Performed six times with similar results. (Bar = 50 μm.)

Figure 2

EPO prevents apoptosis of P19 cells. (A) Cells were serum-deprived for 24 h and apoptotic nuclei were counted after staining with H33258. EPO (1 unit/ml) was added as a 24-h pretreatment (EPO pre) and then removed, or added immediately after serum deprivation (EPO post). (B) Dose–response of the effect of a 24-h pretreatment with EPO on apoptosis induced by subsequent serum and EPO deprivation. (C) Time course of apoptosis induced by serum deprivation in control or 24-h EPO-pretreated cells. (D) Effect of EPO on thymidine incorporation in the presence or absence of serum. (Data represent mean ± SD. *, P < 0.05; **, P < 0.01 indicate significant differences as compared with serum deprivation without EPO treatment.)

Figure 3

Antiapoptotic and neurotrophic effects of EPO in cultured rat motoneurons. (A) Cell viability in mixed neuron-glia cultures in the absence and presence of EPO (10 units/ml) in control cultures and upon serum withdrawal. (B) Cell viability in purified motoneuron cultures in the absence and presence of EPO (10 units/ml) in control cultures and upon serum withdrawal. (C) Cell viability in mixed neuron-glia cultures in the absence and presence of EPO (10 units/ml) in control cultures and upon kainate treatment. (D) Cell viability in purified motoneuron cultures in the absence and presence of EPO (10 units/ml) in control cultures and upon kainate administration. (Data represent mean ± SD. *, P < 0.05 indicates statistical significance as compared with control; •, P < 0.05 denotes statistical significance as compared with EPO during serum withdrawal or kainate treatment.)

Figure 4

EPO induces phosphorylation of tyrosine kinases in rat hippocampal neurons. (A) EPO (0.3 units/ml; 100 pM) and BDNF (100 ng/ml; 3.6 nM) activate Stat5, Akt/PKB, and ERKs in rat hippocampal neurons. Expression of pStat5 (lower band), pAkt/PKB, pERK1 (upper band) and pERK2 (lower band) was monitored by Western blotting. Expression of total ERKs was used as a loading control. The differences seen in total ERKs reflect variations of cell numbers in individual cultures after hypoxic conditions. An increase in the level of activating phosphorylations of Akt/PKB or ERKs relative to total protein content, however, was found to be consistent in three independent experiments. The molecular size of the protein standards are indicated (Left). (B) Inhibition by PD98059 (50 μM) and/or LY984002 (100 μM) of the EPO (0.3 units/ml) or BDNF (100 ng/ml) induced activation of Akt/PKB, ERK1 (upper band), and ERK2 (lower band) in rat hippocampal neurons upon hypoxia.

Figure 5

Inhibition by PD98059 (50 μM) and/or LY984002 (100 μM) of the EPO (0.3 units/ml) induced neuroprotection in rat hippocampal neurons upon hypoxia. ○, level of spontaneous cell death of rat hippocampal neurons under normoxia; •, level of cell death upon 15 h of hypoxia. (Data represent mean ± SD. *, P < 0.05; **, P < 0.01 compared with hypoxia alone; n = 4–5.)